1. Field of the Invention
The present invention relates generally to medical methods and devices. More particularly, the present invention relates to methods, systems, and kits for the delivery of nucleic acids to smooth muscle cells which line the lumen of blood vessels.
A number of percutaneous intravascular procedures have been developed for treating atherosclerotic disease in a patient""s vasculature. The most successful of these treatments is percutaneous transluminal angioplasty (PTA) which employs a catheter having an expansible distal end, usually in the form of an inflatable balloon, to dilate a stenotic region in the vasculature to restore adequate blood flow beyond the stenosis. Other procedures for opening stenotic regions include directional atherectomy, rotational atherectomy, laser angioplasty, stents and the like. While these procedures, particularly PTA, have gained wide acceptance, they continue to suffer from the subsequent occurrence of restenosis.
Restenosis refers to the re-narrowing of an artery within weeks or months following an initially successful angioplasty or other primary treatment. Restenosis afflicts up to 50% of all angioplasty patients and results at least in part from smooth muscle cell proliferation in response to the injury caused by the primary treatment, generally referred to as xe2x80x9chyperplasia.xe2x80x9d Blood vessels in which significant restenosis occurs will require further treatment.
A number of strategies have been proposed to treat hyperplasia and reduce restenosis. Such strategies include prolonged balloon inflation, treatment of the blood vessel with a heated balloon, treatment of the blood vessel with radiation, the administration of anti-thrombotic drugs following the primary treatment, stenting of the region following the primary treatment, and the like. While enjoying different levels of success, no one of these procedures has proven to be entirely successful in treating all occurrences of restenosis and hyperplasia.
Of particular interest, it has recently been proposed to deliver nucleic acids to smooth muscle cells within blood vessels for the treatment of hyperplasia and other disease conditions. See, e.g. U.S. Pat. No. 5,328,470. Progress in vascular gene therapy, however, has been hindered by the limited efficiency and/or toxicity of most currently available transfection materials and techniques. Current methods used to achieve nucleic acid transfer into vascular smooth muscle cells comprise the delivery of naked DNA, cationic liposomes, and specialized adenoviral and retroviral vectors. Each of these approaches are problematic. While the use of adenoviral vectors can achieve relatively high transfection efficiencies, the use of viruses raises concern among many experts in the field.
For these reasons, it would be desirable to provide additional and/or improved methods, systems, kits, and the like for the delivery of nucleic acids to vascular smooth muscle cells and other cells which comprise the vascular wall. It would be particularly desirable if such gene delivery methods were useful for the treatment of hyperplasia in regions of a blood vessel which have previously been treated by angioplasty, atherectomy, stenting, and other primary or secondary treatment modalities for atherosclerotic disease. Such methods should provide efficient gene delivery, result in minimum necrosis of the cells lining the vasculature (particularly smooth muscle cells and endothelial cells), permit targeting of vascular smooth muscle cells, be capable of being performed with relatively simple catheters and other equipment, and suffer from minimum side effects. At least some of these objectives will be met by the invention described hereinafter.
2. Description of the Background Art
Catheters and methods for intravascular transfections are described in U.S. Pat. No. 5,328,470 and published in PCT applications WO 97/12519; WO 97/11720; WO 95/25807; WO 93/00052; and WO 90/11734.
Ultrasound-mediated cellular transfection is described or suggested in Kim et al. (1996) Hum. Gene Ther. 7:1339-1346; Tata et al. (1997) Biochem. Biophy. Res. Comm. 234:64-67; and Bao et al. (1997) Ultrasound in Med. and Biol. 23:953-959. The effects of ultrasound energy on cell wall permeability and drug delivery are described in Harrison et al. (1996) Ultrasound Med. Biol. 22:355-362; Gao et al. (1995) Gene Ther. 2:710-722; Pohl et al. (1993) Biochem. Biophys. Acta. 1145:279-283; Gambihler et al. (1994) J. Membrane Biol. 141:267-275; Bommannan et al. (1992) Pharma. Res. 9:559-564; Tata and Dunn (1992) J. Phys. Chem. 96:3548-3555; Levy et al. (1989) J. Clin. Invest. 83:2074-2078; Feschheimer et al. (1986) Eur. J. Cell Biol. 40:242-247; and Kaufinan et al. (1977) Ultrasound Med. Biol. 3:21-25. A device and method for transfection, endothelial cells suitable for seeding vascular prostheses are described in WO 97/13849.
Local gene delivery for the treatment of restenosis following intravascular intervention is discussed in Bauters and Isner (1998) Progr. Cardiovasc. Dis. 40:107-116 and in Baek and March (1998) Circ. Res. 82:295-305.
A high frequency ultrasonic catheter employing an air-backed transducer which may be suitable for performing certain methods according to the present invention is described in He et al. (1995) Eur. Heart J. 16:961-966. Other catheters suitable for performing at least some methods according to the present invention are described in co-pending application Ser. Nos. 08/565,575; 08/566,740; 08/566,739; 08/708,589; 08/867,007, and 09/223,225, assigned to the assignee of the present invention, the full disclosures of which are incorporated herein by reference.
The present invention comprises methods, systems, and kits for the delivery of nucleic acids to the smooth muscle cells of the type which line coronary arteries and other blood vessels. The delivery of nucleic acids to target cells is generally referred to as xe2x80x9ctransfection,xe2x80x9d and the transfection methods of the present invention are advantageous since they are capable of significantly increasing transfection efficiency, i.e. the amount of nucleic acid materials taken up by the smooth muscle cells to which they are delivered. The methods of the present invention are useful with a wide variety of nucleic acid types. For example, it has been found that significant transfection efficiencies can be obtained even with naked DNA and RNA molecules i.e., nucleic acids which are not incorporated into liposomes, viral vehicles, plasmids, or other conventional nucleic acid vehicles. The methods are not limited to such naked nucleic acids, however, they are also suitable for the delivery of nucleic acids incorporated into liposomes and other vesicles; viral vectors, including both adenoviral vectors and retroviral vectors; plasmids, and the like.
The methods of the present invention are particularly suitable for delivering nucleic acids incorporated into liposomes often referred to as xe2x80x9clipofection,xe2x80x9d to the vascular smooth muscle cells. As is demonstrated in the Experimental section hereinafter, transfection of vascular smooth muscle cells with naked DNA is enhanced significantly by vibratory energy (by a factor of 7.5 in the particular data shown), but overall transfection efficiency still remains at a relatively low level. In contrast, lipofection enhanced with vibratory energy according to the present invention shows a lesser enhancement over lipofection without vibrational energy (by a factor of three in the particular data which are shown), but the overall transfection efficiency, is substantially greater than that which can be achieved with naked nucleic acids, even with vibrational energy enhancement. Thus, the combination of lipofection with vibrational energy enhancement will frequently be preferred. While similar overall transfection efficiencies may be achieved with vibrational enhancement of viral vectors, the use of viral vectors will often not be preferred because of the safety concerns which have been raised with respect to such delivery vehicles. Additionally, as other delivery vehicles are developed as alternatives for variations of the liposome and viral vehicles which presently find use, it will be expected that the vibratory enhancements of the present invention will find use with such methods. A significant advantage of the present invention, however, is that such delivery vehicles are not essential for efficient uptake.
While the methods, systems, and kits of the present invention will preferably be used with in vivo transfection techniques described above, they will also find use with in vitro techniques for transfecting vascular smooth muscle cells in culture. Such in vitro methods will find use in many contexts, such as in the testing of different structural and regulatory genes to determine their effect on vascular smooth muscle cells, the transformation of vascular smooth muscle cells to other predictable phenotypes research purposes, and the like. In other instances, it may be desirable to transfect autologous or heterologous vascular smooth muscle cells in vitro so that the cells can later be xe2x80x9cseededxe2x80x9d back into a patient for a particular therapeutic purpose. For example, vascular smooth muscle cells can be transfected to produce therapeutic proteins which can be released by the transfected cells after they are implanted or otherwise introduced to a patient.
The nucleic acids may be in the form of genes, gene fragments, sense oligonucleotides and polynucleotides, anti-sense oligonucleotides and polynucleotides, and any other type of nucleic acid having biological activity or benefit. Exemplary genes that may be delivered for treating cardiovascular disease and hyperplasia include angiogenic factors, such as vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), tissue inhibitor matrix matallio-proteinase (TIMP), p21, and the like.
Smooth muscle and other vascular cells are transfected according to the present invention by delivering nucleic acids to the cells located, for example, in a target region within a blood vessel or in cell culture. The cells are exposed to vibratory energy at a frequency and intensity selected to enhance the uptake of the nucleic acids by the smooth muscle cells, which line the blood vessel wall. The exposure of the cells to the vibratory energy can occur before exposure or introduction of the nucleic acids, after exposure or introduction of the nucleic acids, or simultaneously with such exposure or introduction. Preferably, exposure of the cells to the vibratory energy will continue for at least a time (total elapsed time) following the introduction or exposure of the cells to the nucleic acids, typically for at least 10 seconds, preferably for at least 60 seconds, more preferably for at least 300 seconds, and still more preferably for at least 900 seconds, usually being in the range from 10 seconds to 900 seconds.
Preferably, the vibratory energy is delivered at a frequency in the range from 1 kHz to 10 MHz, preferably in the range from 20 kHz to 3 MHz, usually from 100 kHz to 2 MHz. The intensity of the vibrational energy will usually be in the range from 20 W/cm2 to 100 W/cm2, preferably in the range from 0.1 W/cm2 to 10 W/cm2, usually from 0.5 W/cm2 to 5 W/cm2. The vibratory energy may be delivered continuously during the transfection event, or alternatively may be delivered intermittently, e.g. with a duty cycle within the range from 1% to 100%, usually from 5% to 95%, preferably from 10% to 50%.
The duration of exposure of the cells to the vibration energy will be a function of total elapsed time (usually within the range and limits set forth above), the duty cycle (percentage of the total elapsed time in which the vibrational energy is turned on), and pulse repetition frequency (PRF; the frequency at which the vibrational energy is turned off and on, typically in the range from 1 Hz to 1000 Hz). Generally, the duty cycle and/or PRF can be controlled to permit heat dissipation to maintain a temperature in the treated artery or cell culture below 45xc2x0 C., preferably below 42xc2x0 C., and more preferably below 40xc2x0 C. Higher temperatures can be deleterious to the viability of the vascular smooth muscle cells.
The vibrational energy will usually be ultrasonic energy and may be delivered in a variety of ways. For example, the vibrational energy may be delivered from an external source, e.g. by focused ultrasonic systems, such as high intensity focused ultrasound (HIFU) systems which are commercially available. Usually, however, the ultrasonic energy will be delivered intravascularly using an interface surface which is disposed within the region within the blood vessel. The interface surface is vibrationally excited to radiate ultrasonic energy directly or indirectly (as defined below) into the blood vessel wall. Typically, the ultrasonic surface is carried on a flexible catheter having a vibrational transducer or other oscillator disposed on the catheter near the surface. The transducer is then energized to vibrate the surface within the desired frequency range and at the desired intensity. Alternatively, ultrasonic or other vibrational energy can be delivered from an external source down a transmission member through the catheter to the interface surface. For in vitro methods, a variety of hand-held probes and transducers could be employed. A particular transducer useful for imparting vibratory energy to cultures of vascular smooth muscle cells is described in the Experimental section hereinafter.
The vibrational energy may be delivered directly into the blood vessel wall, e.g. by contacting the interface surface directly against a portion of the wall within the target region. Alternatively, the vibrational energy can be delivered indirectly by vibrating the surface within blood or other liquid medium within the blood vessel. Usually, the nucleic acids will be released or disposed in the liquid medium. In an exemplary embodiment, the nucleic acids are contained within a suitable transfection medium which is localized within the target region by a pair of axially spaced-apart balloons. The interface surface is also disposed between the balloons, and energy is applied to the entrapped medium via the interface surface. Alternatively, the nucleic acid medium may be delivered to the interior of a porous balloon and/or to fluid delivery conduits secured to the outside of a balloon, where in both cases the vibrational transducer can be mounted on the catheter body within the balloon. Conveniently, the medium containing the nucleic acids can be delivered to the region via the same catheter, optionally being recirculated or replenished via the catheter during the treatment.
Alternatively, the nucleic acids can be delivered to the patient systemically while the vibrational energy is applied locally and/or from an external source as described above.
Optionally, the nucleic acids may be delivered to a vascular target site in the presence of microbubbles of gas or other cavitation nucleation components. It is believed that low intensity vibration of the type preferably employed in the methods of the present invention will generally not induce cavitation in a vascular environment devoid of cavitation nucleii. As cavitation is presently believed to contribute to the formation of pores in the walls of the smooth muscle cells (and thus enhance nucleic acid uptake), the introduction of microbubbles or other cavitation nucleii together with the nucleic acids, e.g. from the same delivery catheter, may significantly enhance the nucleic acid uptake. For example, the nucleic acids may be delivered in a liquid medium to which dissolved gases have been added as cavitation nucleii.
The nucleic acids can be delivered to the smooth muscle or other vascular cells for a variety of purposes. In a preferred example, the nucleic acids are delivered to a region of the blood vessel which has previously been treated by a primary intravascular technique for treating cardiovascular disease, such as balloon angioplasty, directional atherectomy, rotational atherectomy, stenting, or the like. The methods of the present invention for inhibiting intimal hyperplasia in vascular smooth muscle cells will find particular use following stenting procedures in order to prevent or inhibit hyperplasia which can occur following stenting. The nucleic acids delivered will be intended to inhibit hyperplasia and/or promote angiogenesis following such primary treatment. Methods for promoting angiogenesis, of course, need not be performed in conjunction with a primary treatment. Suitable genes for such treatments have been described above.
Kits according to the present invention will comprise a catheter having an interface surface which may be vibrated. The kits will further include instructions for use setting forth a method as described above. Optionally, the kits will further include packaging suitable for containing the catheter and the instructions for use. Exemplary containers include pouches, trays, boxes, tubes, and the like. The instructions for use may be provided on a separate sheet of paper or other medium. Optionally, the instructions may be printed in whole or in part on the packaging. Usually, at least the catheter will be provided in a sterilized condition. Other kit components, such as the nucleic acids to be delivered, may also be included.
Systems according the present invention will comprise a catheter having an interface surface which may be vibrated at the frequencies and power levels described above. Such systems may further include nucleic acids in a form suitable for the transfection or lipofection of vascular smooth muscle cells lining an artery or other blood vessel. The nucleic acids may be naked, viral-associated, but will preferably be incorporated within liposome vesicles in order to enhance transfection efficiency when delivered in the presence of vibratory energy according to the methods of the present invention. The catheter will usually be packaged in a sterile tray, pouch, or other conventional container, while the nucleic acid reagent will be incorporated in an ampoule, bottle, or other conventional liquid pharmaceutical container. Optionally, the catheter and reagent will be packaged together in a box, bag, or other suitable package. Further optionally, the systems may include instructions for use as described above.